Nature | Vol 585 | 24 September 2020 | 567neurons, cranial nerves and dorsal root ganglia, appeared normal in
Δedn3.L+S larvae (Extended Data Fig. 8a–e), subadult Δedn3.L+S frogs
had a mild Hirschsprung disease-like phenotype that included miss-
ing submucosal ganglia and excess goblet cells^39 ,^40 (Extended Data
Fig. 8f–q). Our results show that disrupting Ednrb signalling in lamprey
and frog causes defects in distinct autonomic components of the PNS.
Together with previous work in mammals, these observations imply
that the development of most PNS elements was independent of Edn in
the last common ancestor of lamprey and gnathostomes. They further
suggest that the role of Edn signalling in development of the auto-
nomic nervous system has diverged in tetrapods and/or lamprey. Data
from other key groups—such as ray-finned fishes—should help identify
ancestral and derived roles for Edn signalling in this NCC derivative.
Lamprey Ednrs have dedicated ligands
In vitro binding assays^41 and the similarity of edn and ednr mutant phe-
notypes^7 ,^30 ,^33 suggest that Edn1 is the main ligand for Ednra, whereas
Edn3 is the main ligand for Ednrb. To test whether lamprey Ednra and
Ednrb also have dedicated ligands, we mutated ednA, ednC and ednE, the
only edn genes expressed in tissue-specific patterns during sea lamprey
development^21. Targeting ednC with three different sgRNAs yielded no
reproducible mutant phenotype (see Methods). By contrast, lamprey
ΔednA larvae displayed a combination of heart oedema and skeletal
defects that resembled hypomorphic Δednra individuals, but without
pigmentation defects, whereas ΔednE larvae resembled Δednrb larvae
(Fig. 3l–p, Extended Data Fig. 9). The incomplete loss of melanophores
in ΔednE and Δednrb larvae mimics amniote and teleost edn3 and ednrb1
mutants^10 ,^42 , whereas edn3 mutant salamanders are completely leucis-
tic^43. We noted that, similar to salamanders, a high percentage of Δedn3.
L+S X. laevis exhibited a complete loss of NCC-derived pigmentation
(Extended Data Fig. 7, Supplementary Table 1). We conclude that all
modern vertebrates have an edn that is largely dedicated to ednrb, and
that NCC-derived pigment cell development in modern amphibians is
particularly dependent on Edn3–Ednrb signalling.
Evolutionary history of edn and ednr genes
Despite inconclusive phylogenies^6 ,^21 ,^23 , the similarity of mutant phe-
notypes raise the possibility that lamprey ednA and ednE are cryptic
orthologues of gnathostome edn1 and edn3, respectively. We therefore
used synteny data from the recently completed sea lamprey germline
genome^27 to reevaluate ednr and edn phylogeny. Other than ednra,
our analyses fail to support one-to-one orthology of lamprey and gna-
thostome ednr or edn genes (Extended Data Fig. 10), consistent with
previous reports^5 ,^21 and recent genomic comparisons^44. Although these
analyses leave the precise history of edn and ednr duplication and loss
unresolved, synteny and phylogenetic analyses of flanking genes sup-
port co-orthology of lamprey ednrb, ednE, ednA and gnathostome
ednrb1 and ednrb2, edn1 and edn3, and edn2 and edn4, respectively.
These relationships suggest that duplication of single primordial ednr
and edn genes in the vertebrate stem yielded ednra, ednrb and two edn
genes; the ancestors of the edn1–edn3–ednE and edn2–edn4–ednA
clades. The conserved roles of lamprey EdnE and gnathostome Edn3
further suggest that after this initial ‘1R’ duplication, and possibly after
an additional duplication event, a member of the Edn1–Edn3–EdnE
paralogy group became largely specialized for Ednrb binding.
Later, after the divergence of cyclostomes and gnathostomes,
non-orthologous Edns (EdnA and Edn1) became independently spe-
cialized for Ednra binding in each lineage (Fig. 4f, Extended Data Fig. 10).Conclusions
The origin and early evolution of NCCs has been linked to the rewir-
ing of gene-regulatory networks^1 , the evolution of new genes^3
and genome-wide duplication events^4. Although these are attrac-
tive hypotheses, functional genetic evidence conclusively linking
vertebrate-specific genes and/or gene duplications to NCC evolution
is sparse. This is largely owing to the difficulty of inferring ancestral
gene functions using conventional genetic model organisms, which
represent only a fraction of vertebrate diversity. We compared thefLanceletsHagshesLampreysCartilaginous shesAncestral
ednr-likeTunicatesLoss of ednr-likeBony shes~1,200 living species46 living species~61,000 living species78 living species~1,600 living species30 living speciesa bcdBona de
ednr?
1R2Rrst edn+Head skeletonPigment
Early stem
vertebrateLate stem
vertebrateVertebrate
last common
ancestorAutonomic
PNS derivativesganglia DRGsHeartCyclostomes
(such as lamprey)bcdGnathostomes
(such as Xenopus)aEdnra dependentPre-duplication Ednr dependent
Ednra and Ednrb cooperateEdnrb dependentEdn signalling independentCranialednrand specialization duplicationeAncestral co-opted toednr
neural crest cellsVeradiationrtebrateFig. 4 | The co-option, duplication and specialization of Edn signalling
pathways drove the expansion and diversif ication of NCC subpopulations.
a, Our results suggest that the vertebrate ancestor had bona fide multipotent
NCCs that activated the NCC gene-regulatory network^1 , but developed in the
absence of Edn signalling. b, Before the first whole-genome duplication (1R) in
stem vertebrates, the primordial Edn signalling system was co-opted to NCC,
affecting the patterning and/or proliferation of non-neural NCC derivatives,
but having little effect on the autonomic nervous system. c, Later in the
vertebrate stem, duplication and specialization of the Ednra and Ednrb
signalling pathways resulted in three or four NCC populations with different
Edn signalling requirements, depending on when the cardiac NCC lineage
arose. d, e, Changes to Ednra and Ednrb signalling targets correlate with
divergence of the oropharyngeal skeleton and autonomic nervous system in
the lineages leading to modern cyclostomes (d) and gnathostomes (e).
f, The deduced transitional forms depicted in a–e mapped onto a phylogenetic
tree of extant chordate groups. The colours of the arrows and lines ref lect the
steps of Edn signalling system evolution depicted in a–e. The inferred origin of
edn and ednr, and their duplication during the vertebrate genome-wide
duplications (1R and 2R)^44 , are shown with grey arrows.